The present invention relates to a process for the preparation of electrostatically-linked, aliphatic- or alicyclic-substituted anionic or cationic polysaccharide gels from readily available macroporous ionic polysaccharide chromatographic media. The present invention further relates to the isolation, recovery and purification of non-polar extractives using said polysaccharide gels in a process of hydrophobic interaction chromatography, for the absorption and desorption of the extractives in the presence of and as a result of the concentration and selection of an organic solvent.
Agricultural plants, and waste streams from their processing, by way of an example, may contain components that are now being discovered as having desirable therapeutic and other benefits. For example: some saponins have been shown to exhibit antineoplastic chemotherapeutic value (U.S. Pat. No. 5,558,866), while others find use in the treatment of hypercholesterolemia (U.S. Pat. No. 5,502,038). Still further antifungal (e.g. Crombie, W. M. L., and Crombie, L., Phytochemistry 25: 2069-2073, 1986) and immunogenic (U.S. Pat. No. 5,597,807) activities are known as well as surfactant, emulsifying and foam stabilizing properties which are summarized by Price et al. (CRC Crit. Rev. Food Sci. Nutr., 26, 27-135, 1987). These are but a few examples from the literature.
In addition, some flavones and their glycosides are known to exhibit antimutagenicity (e.g. Peryt, B., et al., Mutation Res. 269: 201-215, 1992), and antitumor activity (e.g. Wei, H., et al., Cancer Res. 50: 499-502, 1990). Further reports of beneficial biological activities and functional properties can be found in a number of reviews (e.g. xe2x80x9cPlant flavonoids in biology and medicine II. Biochemical, cellular, and medicinal propertiesxe2x80x9d Ed. By V. Cody, E. Middleton Jr., J. B. Harborne, and A. Beretz, Liss Inc, New York, 1988.)
Canadian Patent Applications 2,006,957 and 2,013,190 describe ion-exchange processes carried out in aqueous ethanol to recover small quantities of high value by-products from cereal grain processing waste. According to CA 2,013,190, an alcoholic extract from a cereal grain is processed through either an anionic and/or cationic ion-exchange column to obtain minor but economically valuable products. The anionic, cationic and neutral fractions were analysed by thin-layer chromatography and a number of components were identified. For example in an anionic fraction from an alcoholic extraction of hull-less whole oats, the following components were identified: phenolic acids, including ferulic acid, p-coumaric acid and caffeic acid; alkaloids such as avenanthramides; fatty acids, organic acids and amino acids. From the same alcoholic extract the neutral fraction contained compounds, such as: free sugars; phenolics, such as flavonoids; saponins such as avenacosides and desglucosylavenacosides; alkaloids such as the avenacins; and various polar lipids. The compounds identified in the various fractions were not individually isolated by ion-exchange chromatography since many carried the same net charge under the conditions used and thus, this method alone is of little value in the isolation of these useful components for industrial or commercial use. Furthermore, the extractives to be isolated in the present invention are for the most part neutral under conditions used, and thus cannot be isolated by ion exchange chromatography alone, which sorts molecules according to charge.
PCT application WO 92/06710, discloses both the composition and isolation/separation technologies of Quillaja saponins for end uses as immunogenic complexes, using repeated semi-preparative high performance liquid chromatography (HPLC) on a reverse-phase column with an acetonitrile:water gradient elution. The scale of the separation appears not to be intended for production of significant quantities for commercialization but rather for proof of efficacy. The isolated products were produced only on the microgram scale. The scale-up of the separation technique for commercial applications was not disclosed.
U.S. Pat. No. 5,094,960 describes methods of removal of process chemicals from labile biological mixtures by hydrophobic interaction chromatography (HIC) using a resin comprising octadecyl chains coupled to a silica matrix. A method of removing lipid soluble process chemicals such as synthetic detergents, chemical inducers and organic solvents from aqueous biological fluids, particularly directed to producing a protein-containing composition such as blood plasma, cryoprecipitates, and blood plasma fractions, was described. In this disclosure materials and conditions are employed that minimize adsorption and separation of proteins and maximize the removal of the process chemicals. Substantially no biological material is retained on the column. Furthermore, no indication is given as to the intended field of use of any of the compounds and chemicals adsorbed in the process, nor specific conditions to selectively recover any of the adsorbed components retained on the column.
A number of different procedures are known for the isolation and purification of isoflavones. D. E. Walter described a procedure for the preparation of the isoflavone genistin and its aglycone genistein from defatted soybean flakes (J. Amer. Chem. Soc. 63, 3273-3276, 1941). The procedure involved methanol extraction, acetone precipitation, centrifugations and several recrystallizations and gave only one isoflavone, genistin, from which the aglycone genistein could prepared by acid hydrolysis. Ohta et al. described a procedure for isoflavone extraction from defatted soybeans wherein the flakes were extracted with ethanol and the ethanolic extract treated with acetone and ethyl acetate. Column chromatography of the ethyl acetate fraction on silica gel and Sephadex LH-20(trademark) in several additional solvents produced a number of fractions from which individual isoflavones could be recovered by repeated recrystallization (Agric. Biol. Chem. 43: 1415-1419, 1979). Essentially the same separation protocols were used by Farmakalidis, E. and Murphy, P. A. to separate isoflavones extracted using acidified acetone rather than ethanol (J. Agric. Food Chem. 33: 385-389, 1985). These publications are but a few of the many examples in the literature for the laboratory scale extraction and purifications of specific isoflavones. However due to issues of solvent handling and disposal as well as economic feasibility, these procedures are hard to scale up to a commercial process and produce single compounds in undisclosed yields.
U.S. Pat. No. 5,679,806 addressed some of these issues, disclosing a process for the isolation and purification of isoflavones from plant material. The process consisted of three steps whereby the plant material is extracted, the resulting extract fractionated on a reverse phase low pressure polymethacrylate or C18 chromatography column by gradient elution of the adsorbed isoflavones from the column, and finally the resulting fractions containing specific isoflavones are eluted from the column. This process differs in several significant ways from the process described in the embodiments disclosed herein. First, the present process is not restricted to the isoflavone components but also yields a saponin fraction substantially free of isoflavones as well as the entire group of isoflavones which, if desired, can be further fractionated for individual components. Secondly, the present process does not rely on methacrylate or C18-substituted reverse phase inorganic support matrices, which generally display much lower loading capacities and are harder to clean in place than polysaccharide-based gels. Thirdly, the flexibility of the present process allows that conditions be varied, either to capture the isoflavones by absorption or to allow them to elute through the column leaving other non-isoflavone components still absorbed, simply by varying the amount of water in an aqueous alcohol washing solution.
U.S. Pat. No. 5,482,914 teaches that agarose-based gels can be synthesized/modified for the binding of lipoproteins by covalently linking glycidyl ethers of polyoxyethylene detergents of the type HOxe2x80x94(CH2CH2O)nxe2x80x94Oxe2x80x94R to give a modified gel matrix suitable for the removal of lipoproteins from human and animal body fluids. This technology refers only to the chemical processes for producing the gel and makes no claims either for electrostatic binding of ligands such as we describe, or for any examples of separation or recovery from plant material.
Thus, there is still a need for processes, chromatographic procedures and improved absorption media that are adaptable to a wide range of compounds in a commercially viable manner that provide high concentrations of these compounds which can be subsequently recovered in high yield, purity and in unaltered form. There is also a need for a process in which the chromatographic media can be regenerated and re-used many times to reduce both waste disposal costs and replacement costs. Furthermore, for commercial scale production of non-polar extractives it would also be advantageous to reduce the direct contact of solvents such as chlorinated hydrocarbons (e.g. chloroform, dichloromethane), nitrites (e.g. acetonitrile), aromatics (e.g. benzene, toluene), other potentially undesirable reagents (e.g. salts, mineral acids, bases), and chromatographic media contaminants (methacrylate-, divinylbenzene-, styrene-monomers, silica etc.) from direct contact with desired products. To accomplish this latter objective and still achieve the necessary separations, it would be desirable to selectively alter the chromatographic media to achieve the separation required and use only one simple, acceptable solvent, rather than to use a single chromatographic media and rely on a wide range of more unacceptable solvents. It is to these ends that this technology is directed.
The present invention relates to a process for the preparation of electrostatically-linked, aliphatic- or alicyclic-substituted anionic or cationic polysaccharide gels from readily available macroporous ionic polysaccharide chromatographic media.
The present invention also relates to the electrostatically-linked, aliphatic- or alicyclic-substituted anionic or cationic polysaccharide gels so produced.
The present invention further relates to the isolation, recovery and purification of non-polar extractives using said polysaccharide gels in a process of hydrophobic interaction chromatography, for the absorption and desorption of the extractives in the presence of and as a result of the concentration and selection of an organic solvent.
Thus, according to the present invention there is provided a method of preparing an electrostatically-linked, aliphatic or alicyclic-substituted anionic or cationic polysaccharide gel from a macroporous ionic polysaccharide chromatographic matrix comprising the steps of:
attaching, by ion exchange, a hydrophobic ligand containing a strongly ionizable functional group of opposite charge to that of the said polysaccharide, so that a substantial amount of the available ionic sites of the said polysaccharide are occupied by the ligand to form a modified hydrophobic phase component.
Further according to the present invention there is provided an electrostatically-linked, aliphatic- or alicyclic-substituted anionic or cationic polysaccharide gel comprising a hydrophobic ligand electrostatically bonded to a macroporous ionic polysaccharide gel matrix, so that a substantial amount of the available ionic sites of the said ionic polysaccharide gel matrix are occupied by the ligand to form a modified hydrophobic phase component, wherein the ligand contains a strongly ionizable functional group of opposite charge to that of the said ionic polysaccharide gel matrix.
This invention is also directed to a method of isolating a non-polar extractive comprising:
contacting said non-polar extractive in an aqueous organic solvent solution with a gel matrix selected from the group of an electrostatically-linked, aliphatic- or an alicyclic-substituted anionic or cationic polysaccharide gel matrix;
washing said gel matrix with said aqueous organic solvent solution;
washing said gel matrix with additional aqueous organic solvent solution, wherein the proportion of the solvent in said additional solution is increased; and
recovering said extractive from an effluent stream.